Effects of microtubule inhibitors on etoposide accumulation and DNA damage in human K562 cells in vitro.

The effects of microtubule inhibitors on cellular accumulation of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidine-beta-D-glu copyranoside) (VP-16) and subsequent epipodophyllotoxin-induced DNA single-strand breaks were investigated in human leukemia K562 cells. At concentrations of 0.05-20 microM, vinblastine, vincristine, and maytansine similarly increased the steady-state cell concentration of VP-16 (2.5 microM) up to 2-fold. Following removal of extracellular vinblastine, the elevation of cell VP-16 was maintained through an additional 55-min incubation period. Washing cells free of extracellular VP-16 resulted in a nonexchangeable (or bound) component comprising 15-17% of the VP-16 concentration found before removal of extracellular drug. In cells incubated with VP-16 alone, removal of extracellular drug resulted in less than 5% cell retention of drug. At vinblastine concentrations of 0.05-0.2 microM, the increase in cell VP-16 was due to a progressive increase in nonexchangeable VP-16. At greater vinblastine concentrations, up to 10 microM, there was no further increase in nonexchangeable VP-16 but there was a 1.6-fold increase in the exchangeable (or free) concentration of VP-16. Similar elevation of both nonexchangeable and exchangeable VP-16 by 10 microM vincristine and maytansine was observed; however, 50-100 microM podophyllotoxin or taxol was required for comparable elevation of exchangeable drug with no increase of nonexchangeable VP-16. Elevation of exchangeable VP-16 in the presence of vinblastine was due to inhibition of the unidirectional efflux of this epipodophyllotoxin with a 69% decline in the rate constant for efflux. There were no effects of vinblastine on VP-16 influx. There was no enhancement of DNA single-strand break frequency when cells were incubated with 2.5 microM VP-16 and 0.2 microM vinblastine, a concentration of the Vinca alkaloid that increased only nonexchangeable VP-16. VP-16-induced DNA damage was enhanced by vinblastine concentrations above 0.5 microM, concentrations that elevated exchangeable VP-16, with a maximum doubling of radiation equivalent single-strand break frequency observed with 20 microM vinblastine, consistent with the maximum elevation of cell VP-16 with 20 microM Vinca alkaloid. These results indicate that vinblastine and other microtubule inhibitors elevate cell VP-16 by inhibition of the efflux of exchangeable drug and by increasing the level of nonexchangeable drug. Potentiation of VP-16-induced DNA damage is observed only at microtubule inhibitor concentrations which elevate exchangeable VP-16.(ABSTRACT TRUNCATED AT 400 WORDS)